Aging of the erythrocyte. XIX. Decrease in surface charge density of bovine erythrocytes.

Decrease in electrophoretic mobility of erythrocytes, increase in Km of erythrocyte membrane acetylcholinesterase and decrease in the binding constant of 8-anilino-1-naphthalene sulfonate to erythrocyte membranes demonstrate a decrease in surface charge density of bovine erythrocytes during in vivo aging. This phenomenon seems to be species-specific; it may be due to a diminution of the sialic acid content but may also be contributed by conformational changes of membrane proteins.

Simultaneous measurement of triiodothyronine and thyroxine in unextracted serum samples using a double-tracer radioimmunoassay method.

A method is described for the simultaneous measurement of triiodothyronine (T3) and thyroxine T4) in 0.04 ml of unextracted serum. Antibodies were prepared by immunization of rabbits with T3 and T4 conjugated with human serum albumin. Bound and free labeled hormones were separated by the double-antibody technique, and 8-anilino-1-naphthalene sulfonic acid was used to inhibit binding of the two hormones to thyroxine-binding globulin. The validity of the assay using 125I-T3 and 131I-T4 is shown by the excellent recovery of T3 and T4 added to serum and by the finding that curves obtained by assaying various dilutions of a hyperthyroid serum run parallel to the standard curves. In all clinical serum T3 and T4 values obtained using the double-tracer radioimmunoassay method were in excellent agreement with those obtained by single-tracer RIA techniques. The combined T3-T4 method appears to be accurate, sensitive, and specific, thus making the assay highly desirable as a technical time-saver.

Partial characterization of the 1-anilino-8-naphthalene sulfonate-adrenochrome semicarbazide interaction site in erythrocyte ghost membrane fragments.

The effect of adrenochrome semicarbazide on the conformation of erythrocyte ghost membranes has been studied by ANS fluorescence, lipid and sulfhydryl spin labels and circular dichroism. No large conformational alterations in the membrane were detected by these techniques. Noncompetitive quenching of ANS fluorescence by ADCS suggests ADCS to interact with the membrane at sites close to the ANS binding domain.

Phenytoin binding to human albumin.

Binding of phenytoin to human plasma proteins and to human serum albumin is studied using equilibrium dialysis method at pH 7.4 and 37 degrees C. Phenytoin is mainly bound to albumin, the percentage of bound drug being constant over a wide range of total drug concentrations. Calculation of the drug binding parameters show a low affinity, k = 745 M-1, and a high number of binding sites, n = 8. Palmitic acid and some acidic drugs, warfarin and phenylbutazone added to human serum albumin, decreased phenytoin binding in a non competitive way. Basic and non-ionizable drugs, on the other hand, did not modify phenytoin binding.

[Changes in the serum albumin system during toxic syndrome and their pharmacologic correction]. / Izmeneniia sistemy syvorotochnykh al'buminov pri toksicheskom sindrome i ikh farmakologicheskaia korrektsiia.

In connection with the important role of serum albumins in pathogenesis and sanogenesis of numerous toxic states, we examined binding capacity of these proteins and conditions of their binding sites after acute poisoning with tetrachloromethane (TCM) and administration of antihypoxic agents (sodium gamma-hydroxybutirate), antioxidants (Dibunol), and actoprotector (Tomersol) to rats. We demonstrated that tetrachloromethane intoxication (3.2 g/kg over 24 h) was accompanied by a certain decrease (by 13.8%) in the total blood serum level of albumins and a tendency to a decrease in the value of the binding constant of negatively charged fluorescent probe 1-(phenylamino)-8-sulfonaphthalene. Under these conditions, the mean number of probe binding sites per albumin molecule increases, and as a result, the total concentration of albumin binding sites in the serum remains virtually unchanged. We found that accessibility of the probe to a quenching agent (potassium nitrate) increases in the protein--probe complex under intoxication conditions, suggesting that the type of interaction between the protein and the fluorescent probe changes as well. Therapeutic/prophylactic administration of an antioxidant, antihypoxic agent, or actoprotector leads to an increase in the level of albumin in the serum (Tomersol), partial normalization of its binding properties (binding constant in the case of sodium gamma-oxybutirate, mean number of binding sites per molecule for Dibunol and Tomersol), and the state of binding sites (sodium gamma-oxybutirate, Dibunol).

Binding study of the fluorescence probe 1-anilino-8-naphthalensulfonate to human plasma and human and bovine serum albumin using potentiometric titration.

The binding of 1-anilino-8-naphthalenesulfonate (ANS) to bovine serum albumin (BSA), human serum albumin (HSA), and human plasma has been studied by potentiometric titration utilizing a laboratory constructed ion selective electrode (ISE) of ANS. Three classes of ANS binding sites were found on BSA, HSA, and plasma at 25 and 37 degrees C. Computer analysis of the data resulted in estimates for the association constants, number of binding sites (HSA, BSA), and binding capacity of each class. The association constants for the first class of binding sites at 25 degrees C were found to be 7.53 (+/- 0.59) x 10(5), 2.70 (+/- 0.20) x 10(5), and 2.64 (+/- 0.26) x 10(5) M-1 for BSA, HSA, and plasma, respectively. Lower values for the association constants of all binding classes were estimated at the higher temperature (37 degrees C). The binding capacity for ANS decreased in the order BSA, plasma, HSA.

1-Anilino-8-naphthalene sulfonate binding site on human erythrocyte membrane using fluorescence lifetime and polarization.

It was shown that the human erythrocyte ghost membrane had two kinds of binding site for 1-anilino-8-naphthalene sulfonate (ANS) from binding kinetics. In measuring the fluorescence lifetime of ANS in the human erythrocyte ghost membrane suspension, two kinds of fluorescence lifetime were obtained: tau 1 = 15 ns at low ANS concentration and tau 2 = 8.4 ns at high ANS concentration. Comparing the results obtained from binding kinetics with those from fluorescence lifetime, it was considered that more hydrophobic binding site with fluorescence lifetime tau 1 contained the binding site with high and low binding constant and less hydrophobic binding site with fluorescence lifetime tau 2 corresponded to the binding site with low binding constant. From the results of binding kinetics of ANS to the erythrocyte membrane and the extracted membrane components (proteins and lipids), and those of the rotational relaxation time obtained from the ANS polarization in the erythrocyte membrane suspension, it was shown that the binding sites of ANS in the human erythrocyte ghost membrane were mainly composed of proteins at low ANS concentrations and lipids at high ANS concentrations.

Effect of heparin injection on plasma protein binding of 1-anilino-8-naphthalenesulfonate and salicylate in rats.

After intravenous injection of heparin, the plasma protein binding of 1-anilino-8-naphthalenesulfonate (ANS) was remarkably decreased in rats. This effect occurred within one min after the injection of 1000 units/kg of heparin and lasted for about 30 min. The change in the binding of ANS was closely related to the plasma concentration of free acids (FFA), which was suggested as one of the heparin-induced inhibitors. The free fatty fraction of salicylate in plasma after the intravenous injection of heparin, has a pronounced variation, and also had a statistically significantly correlation with the plasma free fraction of ANS. It was suggested that ANS might be useful for the prediction of the heparin-induced changes in the plasma protein binding of acidic drugs.

Kinetic analysis of the dose-dependent hepatic handling of 1-anilino-8-naphthalene sulfonate in rats.

The dose dependency in the hepatic transport of an anionic fluorescent dye, 1-anilino-8-naphthalene sulfonate (ANS), was investigated by measuring the plasma disappearance and biliary excretion in rats. Bulk of the administered ANS distributed into the liver at 10 min after iv bolus injection. The plasma disappearance curves of ANS were then kinetically analyzed based on a two-compartment model, in which the ligand is eliminated only from the peripheral compartment (liver compartment). The total body clearance (CLtot) decreased with increasing dose of ANS. That is, the values of CLtot were 4.06 and 1.98 ml/min/per kg at the doses of 3 and 100 mumol/kg, respectively. The clearances of the uptake and sequestration processes (CLup and CLseq, respectively) for a total ligand were constant irrespective of dose, while the efflux clearance (CLeff) for a total ligand was increased by twofold with increasing dose. A mechanism for the increase in the CLeff value might be explained by a saturation of the ANS binding to the intracellular proteins. The hepatocellular distribution and the binding of ANS to cytosolic proteins were then determined. ANS mainly distributed to the cytosol fraction, and the unbound fraction in the cytosol increased from approximately 0.04 to 0.09 when the cytosolic concentrations of ANS increased from 40 to 900 microM, respectively. In spite of such increase in the unbound fraction in the cytosol, the CLseq values remained unchanged with increasing dose, suggesting that the saturation of sequestration clearance for unbound ANS might occur. Furthermore, the plasma disappearance curves of ANS at various doses were simultaneously analyzed based on three nonlinear kinetic models: Model I is a model incorporating both saturable intracellular binding and saturable sequestration; Model II is a model incorporating only saturable intracellular binding; Model III is the model incorporating only saturable sequestration. Goodness-of-fit evaluated by AIC value was best for Model I. Taken together, the nonlinearity in the plasma clearance of ANS was confirmed to be attributed to saturation of both its binding to cytosolic proteins and sequestration process.

Effect of various organic anions on the plasma disappearance of 1-anilino-8-naphthalene sulfonate.

The effects of various organic anions on the hepatic transport of an anionic fluorescent dye, 1-anilino-8-naphthalene sulfonate (ANS) were investigated by measuring the plasma disappearance-time profiles in rats. Ten min after the i.v. administration of ANS (3 mumol/kg), various organic anions (60 mumol/kg) were injected in a bolus. Sulfobromophthalein (BSP), bromophenol blue (BPB) and rose bengal (RB) induced a transient increase in the plasma concentration of ANS (the so-called 'counter-transport' phenomena). The effect of rose bengal was somewhat different. After the administration of rose bengal, the plasma concentration of ANS decreased rapidly followed by a gradual increase. On the other hand, after the administration of bilirubin and taurocholate, the transient increases in plasma ANS concentrations were minimal. No effect was observed after the administration of phenolsulfophthalein (PSP) or oleate. The effects of these organic anions on the binding of ANS to rat liver cytosols were examined by equilibrium dialysis. Sulfobromophthalein, bromophenol blue and rose bengal, which yielded an in vivo 'counter-transport' phenomena, markedly inhibited ANS binding to cytosolic proteins. On the other hand, the other organic anions examined had very small, if any, inhibitory effect. The ANS binders in the cytosol were then identified by gel filtration. ANS bound mainly to X and Y (ligandin) fractions in the cytosol. Sulfobromophthalein, which is one of the organic anions exhibiting the in vivo 'counter-transport' phenomenon, remarkably inhibited ANS binding to ligandin fraction. It was thus suggested that the in vivo 'counter-transport' phenomena may be also explained by the enhancement of back diffusion due to the displacement of intracellular binding. In conclusion, one should be more cautious in interpreting data obtained from so-called in vivo 'counter-transport' experiments.

Cystic fibrosis--V. Does cystic fibrosis alter the values of dynamic parameters of erythrocyte membrane ghosts?

1. The dynamic properties of erythrocyte membranes in CF children have been investigated by means of fluorescence and ESR techniques. 2. It has been revealed that the apparent distance separating the membrane protein tryptophan and bound 1-anilino-8-naphthalenesulphonate (ANS) molecules is decreased in CF children which results in a significant increase of the maximum energy transfer efficiency. 3. The slight increase in the ratio hw/hs of maleimide bound to membrane protein-SH groups of erythrocytes in cystic fibrosis may ensue the lowered membrane protein immobilization in the plane of lipid bilayer, especially at the intrinsic, more slowly reacting thiol groups.